Synthesis and biological evaluation of novel fused triazolo[4,3-a] pyrimidinones

Article abstract of DOI:10.3906/kim-1501-144

The reaction of thione 3 or its 2-methylthio derivative 4 with hydrazonoyl halides 5a-l, in the presence of triethylamine, yielded the corresponding triazolo[4,3- a]pyrimidin-5(1H)-ones 8a-l. The structure of compounds 8a-l was further confirmed by the re

Full text of DOI:10.3906/kim-1501-144

Turk J Chem
Turkish Journal of Chemistry
(2015) 39: 510 – 531
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1501-144
Research Article
Synthesis and biological evaluation of novel fused triazolo[4,3-a] pyrimidinones
Ikhlass ABBAS1, Sobhi GOMHA1,∗, Mohamed ELNEAIRY1,
Mahmoud ELAASSER2, Bazada MABROUK1
1Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt
2Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt
Received: 30.01.2015
•
Accepted/Published Online: 02.04.2015
•
Printed: 30.06.2015
Abstract: The reaction of thione 3 or its 2-methylthio derivative 4 with hydrazonoyl halides 5a–l, in the presence of
triethylamine, yielded the corresponding triazolo[4,3-a]pyrimidin-5(1H)-ones 8a–l. The structure of compounds 8a–l
was further confirmed by the reaction of 3 with the appropriate active chloromethylenes 11a–c followed by coupling of
the products with benzenediazonium chloride to afford the azo-coupling products 6b, f, and j, which were converted in
situ to 8b, f, and j. 2-Hydrazinyl-pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin-4(3H)-one (13) was prepared and condensed
with different aldehydes 14a–f to give the corresponding hydrazone derivatives 15a–f. Oxidative cyclization of the
hydrazones 15a–f give the corresponding triazolo[4,3-a] pyrimidin-5(1H)-one derivatives 16a–f.
The antimicrobial activity of the products was evaluated and the results revealed that compounds 8f and 15f
showed strong activity against gram-positive bacteria while compound 15d showed the highest activity against gram-
negative bacteria. Moreover, compounds 15b, 8d, 8e, 8c, 8l, and 8j exhibited significant antifungal activity. In
addition, the antitumoral activity of the synthesized products against different cancer cell lines was determined and the
results revealed that compound 12c was the most active against MCF-7, HepG-2, HCT-116, and HeLa with IC50 values
of 0.51, 0.72, 0.95, and 0.95, respectively, as compared with doxorubicin as positive control.
Key words: Triazolopyrimidinones, cyclizations, hydrazonyl chlorides, antimicrobial, anticancer activity
1. Introduction
It is known that cancer is one of the most dangerous diseases, caused by uncontrolled growth and spread of
abnormal cells, initiated by viruses, smoking, chemicals, or diet.1 Cancer can lead to death if left untreated.
Therefore, many of the research efforts aim to develop new anticancer drugs.2−6
The 1,2,4-triazolopyrimidines have attracted growing interest due to their important pharmacological
activities, such as antitumor potency,7−12 antimalarial,13 antimicrobial,14−17 anti-inflammatory,18 inhibition
of kinase insert domain containing receptor (KDR kinase),19 antifungal,20 and macrophage activation.21 In
addition, triazolo[4,3-a]pyrimidine derivatives were reported to be useful as antihypertensive,22 anxiolytic,23
and cardiovascular24,25 agents.
In view of these reports and in continuation of our previous work on the synthesis of bioactive heterocyclic
compounds,26−32 we were interested in the synthesis of new fused triazolopyridinones to investigate their
antimicrobial and cytotoxic potential activities.
∗Correspondence: s.m.gomha@hotmail.com
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2. Results and discussion
2.1. Chemistry
The starting compound 7,9-bis(4-methoxyphenyl)-2-thioxo-2,3-dihydropyrido[3’,2’:4,5] thieno[3,2-d]pyrimidin-
4(1H)-one (3) was prepared by adopting a reported procedure33 as depicted in Scheme 1. Thus, the reaction
of 4,6-di(4-methoxyphenyl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (1)34 with chloroacetamide in refluxed
ethanol containing KOH afforded compound 2. The structure of compound 2 was confirmed on the basis of
its spectral data (see Experimental). Treatment of the latter compound with CS2 in pyridine followed by
acidification led to the formation of the starting material 3. The structure of 3 was evidenced by its spectral
data (mass, IR, 1 H NMR) and thermal analysis. Its IR spectrum revealed the absorption bands of two –NH
and CO in the regions 3459, 3359, and 1691 cm−1 , respectively while its 1 H NMR spectrum showed two
characteristic signals at δ= 9.29 and 12.99 ppm (D2 O exchangeable) assignable to two –NH protons.
Scheme 1. Synthesis of thione 3 and its methylthio derivative 4.
The methylthio derivative 4 was prepared from the reaction of thione 3 with methyl iodide in the presence
of anhydrous K2 CO3 . The 1 H NMR spectrum of compound 4 showed the signals of S–CH3 and NH at δ=
3.56 and 12.96 ppm, respectively.
Reaction of 3 with each of 5a–l was carried out in dioxane, in the presence of triethylamine, under
reflux until hydrogen sulfide ceased to evolve, affording, in each case, one isolable product as evidenced by TLC
analysis (Scheme 2). The isolated products were assigned the structure of pyridothieno[3,2-d]triazolo-pyrimidin-
5(1H)-ones 8a–l rather than its isomeric structure pyridothieno[2,3-e]triazolo-pyrimidin-5(3H)-one 10 based
on their elemental analyses and IR, 1 H NMR, and 13 C NMR spectra (see Experimental). For example, the
δ values (δ = 164 ppm) for the carbonyl carbon signal in the 13 C NMR spectra of 8a are similar to those of
compounds of type A (δ = 161–164 ppm) and different from those of their isomers having structure B (δ =
170–175 ppm)35 (Figure 1). This finding excludes structure 10 for the products.
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Scheme 2. Synthesis of pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-ones (8a–l).
Compound 8 was further evidenced by alternate synthesis, via refluxing 2-methylthio derivative 4 with
5a–l in the presence of triethylamine until the evolution of methanethiol ceased and products formed that
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proved identical in all respects (IR, MS, mp, and mixed mp) with 8a–l. The assumption that the latter reaction
proceeds through the amidrazone intermediate 9 is compatible with the literature.36
13
Figure 1. 13 C NMR shifts of strategic carbon atoms.
Formation of compounds 8 could be started with the formation of thiohydrazonate esters 6 from
hydrazonoylation of thione 3, followed by Smiles rearrangement37 to form the respective thiohydrazides 7.
Cyclization of 7 followed by removal of H2 S yielded 8 (Scheme 2). Alternatively, the formation of 8 from
methylthio derivative 4 and hydrazonoyl halides 5 can be accomplished via cyclization of the amidrazone 9
(Scheme 2).
The involvement of 6 and 7 as intermediates in the formation of 8 was evidenced by alternate synthesis
of 8b, f, and j (Scheme 2). Thus, treatment of 3 with each of the active chloromethylene compounds 11a–c
in KOH/DMF at room temperature yielded the substitution products 12a–c (Scheme 3). The structure of the
latter products was elucidated by its microanalysis and spectral data (mass, IR, 1 H NMR). The 1 H NMR data
showed singlet signals at δ = 2.34 and 4.01 ppm assignable to the methyl and methine protons, respectively, in
addition to the characteristic signals corresponding to COMe, COOEt, and CONHPh groups in the compounds
12a–c, respectively. The formation of 12a–c from the reaction of 3 with 11a–c (Scheme 3) is analogous to
S-alkylation reactions reported for 2-thioxopyrimidines.38
Coupling of 12a–c with benzenediazonium chloride yielded the corresponding thiohydrazonate esters 6b,
f, and j, which undergo in situ Smiles rearrangement to give the intermediates 7b, f, and j, which could be
cyclized into the corresponding 8b, f, and j. This finding indicates that 6 and 7 are intermediates in the studied
reactions of 3 with 5a–l.
Finally, the suggestion that the site of cyclization of the thiohydrazide intermediates 7 involves N-3 to
give 8 is consistent with literature reports.39−41
2-Hydrazinyl-7,9-di(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin-4(3H)-one (13) was prepared
by refluxing compound 4 with NH2 NH2 .H2 O in DMF. Condensation of compound 13 with different aldehydes
14a–f in acetic acid gave the corresponding hydrazone derivatives 15a–f (Scheme 4). The mass spectra of
the isolated products 15a–f showed the molecular ion peaks at the expected m/z values. Their IR spectra
showed the disappearance of the NH2 group, and revealed in each case a carbonyl band in the region 1651–1670
cm−1 and two bands at 3444–3448 and 3348–3363 cm−1 assignable to two –NH groups. Furthermore, the 1 H
NMR spectra showed, in each case, the presence of the azomethine and two –NH protons at δ = 8.11–8.20,
10.98–11.16, and 11.69–11.89 ppm, respectively.
Oxidative cyclization of the hydrazone derivatives 15a–f with bromine in acetic acid in the presence of
sodium acetate at room temperature yielded in each case one isolable product 16a–f (Scheme 4). The products
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Scheme 3. Alternative synthesis of pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-ones (8b, f, and
j).
16a–f were deduced from their spectral data (IR, 1 H NMR, and ESI-MS) and elemental analyses, which are
listed in the Experimental part. In addition, compound 16a was synthesized by an alternative method via
reaction of compound 13 with benzoyl chloride in pyridine.
3. Biological activity
3.1. Antimicrobial evaluation
The newly synthesized target compounds were evaluated for their in vitro antibacterial activity against two
gram-positive bacterial species (Bacillus subtilis and Staphylococcus aureus), two gram-negative bacterial species
(Escherichia coli and Pseudomonas aeruginosa), two moulds (Aspergillus fumigatus and Syncephalastrum
racemosum), and two yeasts (Candida albicans and Geotrichum candidum) using a modified well diffusion
method. The organisms were tested against the activity of solutions of concentrations (5 mg/mL) and using
inhibition zone diameter in millimeters as criterion for the antimicrobial activity (agar well diffusion method).
The results of testing for antibacterial and antifungal effects are summarized in Tables 1 and 2. As shown
by these results, the new fused triazolopyrimidinone derivatives tested displayed variable in vitro antibacterial
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Scheme 4. Synthesis of pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (16a–f).
and antifungal actions. In general, the chemical structure of the whole molecule, comprising the nature of the
heterocyclic system as well as the type of the substituted function present in the heterocyclic ring structure,
has a pronounced effect on antimicrobial activity. Most of the corresponding substituted analogues produced
higher inhibitory effects against bacteria similar or superior to the reference drug tetracycline.
From the screening results, it can be seen that compounds 8f and 15f showed the highest activity against
gram-positive bacteria and compound 15d showed the highest activity against gram-negative bacteria. The rest
of the compounds showed good to moderate activity against the tested bacteria compared with the standard
drugs.
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Table 1. In vitro antibacterial activity of the tested compounds by well diffusion agar assay expressed as inhibition
zone diameter (mm) in the form of mean ꢀ SD.
Gram-positive bacteria
Gram-negative bacteria
Tested
Bacillus
subtilis
Staphylococcus Escherichia Pseudomonas
compounds
aureus
coli
aeruginosa
13.2 ꢀ 0.4
11.6 ꢀ 0.9
8.1 ꢀ 0.6
3
17.9 ꢀ 0.5 22.8 ꢀ 0.8
18.2 ꢀ 0.7 22.4 ꢀ 0.4
11.9 ꢀ 0.3 17.2 ꢀ 0.6
19.7 ꢀ 0.8 24.7 ꢀ 0.7
18.5 ꢀ 0.9 25.6 ꢀ 1.3
21.7 ꢀ 0.8 24.1 ꢀ 0.9
30.8 ꢀ 1.2 26.8 ꢀ 0.7
19.2 ꢀ 0.4 25.6 ꢀ 0.8
18.3 ꢀ 0.6 23.9 ꢀ 0.6
13.4 ꢀ 0.2 9.7 ꢀ 0.3
19.1 ꢀ 0.5 12.8 ꢀ 0.2
18.7 ꢀ 0.8 17.2 ꢀ 0.7
23.6 ꢀ 0.7 25.9 ꢀ 1.3
12.1 ꢀ 0.8 9.7 ꢀ 0.4
21.7 ꢀ 0.6 17.9 ꢀ 0.8
15.3 ꢀ 0.5 21.8 ꢀ 1.1
21.8 ꢀ 0.9 25.6 ꢀ 0.8
20.2 ꢀ 0.7 18.9 ꢀ 0.5
14.2 ꢀ 0.4 19.7 ꢀ 0.9
21.3 ꢀ 0.7 25.3 ꢀ 1.1
13.1 ꢀ 0.5 19.8 ꢀ 0.8
29.3 ꢀ 1.1 24.3 ꢀ 0.7
19.3 ꢀ 0.7
18.1 ꢀ 0.9
10.6 ꢀ 0.3
18.2 ꢀ 0.7
14.7 ꢀ 0.6
22.3 ꢀ 1.1
24.6 ꢀ 0.9
20.1 ꢀ 0.8
10.7 ꢀ 0.4
21.5 ꢀ 0.9
22.3 ꢀ 1.1
20.4 ꢀ 0.8
23.8 ꢀ 0.7
7.2 ꢀ 0.3
15.8 ꢀ 0.4
17.6 ꢀ 0.6
24.1 ꢀ 0.9
10.8 ꢀ 0.5
10.9 ꢀ 0.4
30.7 ꢀ 1.3
10.2 ꢀ 0.6
19.7 ꢀ 0.8
30.2 ꢀ 0.6
8a
8b
8c
13.4 ꢀ 0.7
10.3 ꢀ 0.5
17.2 ꢀ 0.9
18.4 ꢀ 0.8
13.3 ꢀ 0.7
8.5 ꢀ 0.4
8d
8e
8f
8g
8h
8i
13.7 ꢀ 0.2
16.6 ꢀ 0.9
12.8 ꢀ 0.6
12.3 ꢀ 0.4
7.4 ꢀ 0.3
8j
8k
8l
12a
12b
12c
15a
15b
15c
15d
15e
15f
14.1 ꢀ 0.8
11.4 ꢀ 0.5
16.9 ꢀ 0.7
14.7 ꢀ 0.3
7.6 ꢀ 0.4
24.6 ꢀ 0.9
11.5 ꢀ 0.4
13.6 ꢀ 0.6
27.4 ꢀ 0.8
Tetracycline 28.7 ꢀ 0.5 26.4 ꢀ 0.7
Interestingly, compound 15b showed higher inhibitory activity against Aspergillus fumigates compared
with amphotericin B reference drug. Despite promising in vitro antifungal activity of some of the newly
synthesized compounds, only compounds 8d, 8e, 8c, 8l, and 8j among the compounds tested exhibited high
antifungal activity as compared with those of the reference drug against yeast species. Compound 8c also
exhibited a higher inhibitory effect than the reference drug, amphotericin B.
The mean values of the inhibition zone diameter obtained for these compounds suggest that all synthesized
compounds possess significant antimicrobial activity against most test organisms used in these assays (Tables 1
and 2); therefore, minimum inhibitory concentration (MIC) of various synthesized compounds was evaluated in
vitro using the two-fold serial dilution technique, while the lowest concentration showed no growth as the MIC.
The fungicides amphotericin B and gentamicin as well as the bactericides ampicillin and tetracycline were used
as references to evaluate the potency of the tested compounds under the same conditions. The results of MIC
are reported in Table 3.
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Table 2. In vitro antifungal activity of the tested compounds by well diffusion agar assay expressed as inhibition zone
diameter (mm) in the form of mean ꢀ SD.
Filamentous fungi
Yeasts
Tested
Aspergillus Syncephalastrum Candida
Geotrichum
candidum
-
compounds
fumigatus
racemosum
albicans
3
11.4 ꢀ 0.5 8.4 ꢀ 0.9
12.1 ꢀ 0.7 10.3 ꢀ 0.5
-
8a
10.4 ꢀ 0.9 8.5 ꢀ 0.3
8b
8c
-
-
-
-
26.5 ꢀ 0.8 24.2 ꢀ 0.9
29.3 ꢀ 1.2 18.9 ꢀ 0.5
23.1 ꢀ 0.9 19.2 ꢀ 0.8
11.7 ꢀ 0.8 8.1 ꢀ 0.4
26.2 ꢀ 2.1 21.8 ꢀ 0.9
24.6 ꢀ 0.7 18.3 ꢀ 0.6
26.4 ꢀ 1.3 20.9 ꢀ 0.8
25.2 ꢀ 0.8 24.3 ꢀ 0.9
24.5 ꢀ 0.5 22.6 ꢀ 1.1
14.7 ꢀ 0.9 13.2 ꢀ 0.7
18.3 ꢀ 0.6 14.5 ꢀ 0.3
17.7 ꢀ 1.1 14.1 ꢀ 0.7
11.5 ꢀ 0.4 9.2 ꢀ 0.5
22.3 ꢀ 0.9 16.3 ꢀ 0.8
21.7 ꢀ 0.8 17.6 ꢀ 0.9
23.2 ꢀ 1.2 20.2 ꢀ 0.7
8d
8e
8f
8g
8h
8i
-
-
8j
18.9 ꢀ 0.6 12.7 ꢀ 0.4
26.5 ꢀ 1.1 20.3 ꢀ 0.8
28.3 ꢀ 0.8 18.2 ꢀ 0.5
8k
8l
12a
12b
12c
15a
15b
15c
15d
15e
15f
-
-
-
-
8.9 ꢀ 0.5
9.3 ꢀ 0.7
-
-
11.3 ꢀ 0.7 8.8 ꢀ 0.5
9.7 ꢀ 0.4
8.4 ꢀ 0.5
8.9 ꢀ 0.4
7.1 ꢀ 0.9
-
-
29.8 ꢀ 1.2 22.3 ꢀ 0.6
12.2 ꢀ 0.6 8.7 ꢀ 0.5
18.2 ꢀ 0.7 14.5 ꢀ 0.4
20.6 ꢀ 1.3 18.5 ꢀ 1.1
9.2 ꢀ 0.6 7.6 ꢀ 0.8
11.3 ꢀ 0.7 10.1 ꢀ 0.9
-
-
-
-
11.4 ꢀ 0.5 9.3 ꢀ 0.3
9.8 ꢀ 0.8
7.6 ꢀ 0.3
Amphotericin B 27.1 ꢀ 0.6 23.2 ꢀ 0.9
17.9 ꢀ 0.3 19 ꢀ 0.5
Compound 15d showed an appreciable broad spectrum of action against both gram-positive and gram-
negative bacteria with an antibacterial potency higher than that of their reference drug. Compound 15d reached
the highest potency with MIC 100 and 50 µg/mL against S. aureus, E. coli, and P. aureginosa, respectively.
Significant MIC values were determined for compounds 8c and 15b against the tested fungi. Based on the
biological evaluation, most of the compounds tested, in particular 8c, 8f, 8l, 15b, and 15f, may be considered
new antimicrobial agents.
3.2. Cytotoxic activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with
5-fluorouracil, doxorubicin, and imatinib42 as standard drugs. The data generated were used to plot a dose
response curve of which the concentration of test compounds required to kill 50% of the cell population (IC50
)
was determined and the results revealed that all the tested compounds showed inhibitory activity to the tumor
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Table 3. Antimicrobial activity minimum inhibitory concentration (MIC µg/mL) of synthesized compounds compared
with standard drugs.
Minimum inhibitory concentration (µg/mL)
Tested
Gram +ve bacteria Gram –ve bacteria Fungi
compounds
B.
S.
E.
P.
A.
S.
C.
G.
subtilis
500
100
500
150
200
200
100
150
200
250
200
200
125
500
250
250
150
150
250
125
500
100
50
aureus
500
100
250
150
125
150
150
125
150
500
500
200
125
500
250
150
125
150
250
100
250
100
50
coli
500
250
500
200
250
200
150
200
aeruginosa fumigatus racemosum albicans candidum
3
1000
500
500
250
500
250
200
250
1000
500
-
1000
1000
-
-
-
8a
1000
-
1000
-
8b
8c
125
100
150
500
125
150
-
150
250
200
1000
250
150
-
150
150
150
500
500
250
1000
250
200
200
-
150
150
250
500
500
250
1000
250
250
250
-
8d
8e
8f
8g
8h
1000 1000
8i
200
125
250
125
500
250
500
500
8j
150
125
100
-
500
150
250
-
8k
8l
12a
1000 1000
12b
250
150
125
250
250
50
250
250
250
250
500
50
500
250
500
50
500
500
1000
100
500
250
-
-
-
12c
500
-
500
-
15a
15b
125
500
250
-
250
500
250
-
15c
250
200
-
15d
15e
500
200
50
500
250
50
15f
500
-
500
-
500
-
500
-
Tetracycline
Ampicillin
Griseofulvin
Amphotericin B
100
-
250
-
100
-
100
-
-
-
-
-
100
50
250
100
250
100
200
100
-
-
-
-
cell lines in a concentration dependent manner. Cytotoxic activity was expressed as the mean IC50 of six
replicates in three independent experiments. The results are represented in Table 4 and Figures 2–4 show that
compounds 12a, 12b, and 12c have the highest cytotoxic activity against the two tumor cell lines MCF-7
and HepG-2, compared with reference drugs; thus these compounds were evaluated for their inhibitory effect
on HCT-116 and HeLa cell lines. Moreover, compound 12c was the most active against MCF-7, HepG-2,
HCT-116, and HeLa with IC50 values of 0.51, 0.72, 0.95, and 0.95, respectively, as compared with doxorubicin.
Interestingly, compounds 12a, 12b, 12c, 15a, and 15c exhibited 48- to 1.07-fold more potent antitumor activity
than imatinib against breast carcinoma (MCF-7) cell lines and were the most active among their analogues.
Furthermore, compounds 15a, 8f, and 15c showed cytotoxic effects against HepG2 comparable to imatinib.
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Table 4. The in vitro inhibitory activity of tested compounds against tumor cell lines expressed as IC50 values (µg/mL)
ꢀ standard deviation from six replicates.
Tumor cell lines
Tested compounds
MCF-7 HepG2 HCT-116 HeLa
3
48.5
44.9
32.5
37.9
29.9
36.4
35.3
39.7
46.7
36.4
42.8
26.1
24.9
3.5
> 50
41.0
32.8
37.9
35.1
38.4
24.7
40.2
32.0
41.1
36.6
35.7
28.5
5.0
-
-
8a
-
-
8b
-
-
8c
-
-
8d
-
-
8e
-
-
8f
-
-
8g
-
-
8h
-
-
8i
-
-
8j
-
-
8k
-
-
8l
-
-
12a
9.3
11.6
12b
3.8
4.0
10.9
11.8
12c
0.51
23.1
41.6
20.3
37.8
34.6
24.7
0.46
24.6
3.9
0.72
26.7
44.9
24.0
37.8
37.9
27.9
0.42
18.9
4.6
0.95
0.95
15a
-
-
15b
-
-
15c
-
-
15d
-
-
15e
-
-
15f
-
-
Doxorubicin
Imatinib
5-Fluorouracil
0.46
9.7
4.3
0.63
34.1
6.8
Moreover, compounds 8a, 8b, 8c, 8d, 8e, 8g, 8h, 8i, 8j, 8k, 15b, 15d, and 15e were less active than
imatinib.
In light of the results presented in this work and taking into account that this preliminary study did not
produce conclusive evidence regarding a structure antimicrobial activity relationship, we focused our attention
on the most promising compounds, 8c, 8f, 8d, and 15d, as an interesting starting point for the development
of a new class of antimicrobial agents. However, compounds 12c, 12a, and 12b exhibited promising inhibitory
activity against the four tested tumor cells. We think that research in this direction should be encouraged in
order to broaden the applicability of these new heterocyclic frameworks to serve as leads for designing novel
chemotherapeutic agents.
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8f
120
8e
8d
8c
8b
8a
3
Doxorubicin
5-FU
8g
8h
8i
8j
8l
Imatinib
120
100
80
60
40
20
0
HepG 2
HepG2
100
80
60
40
20
0
0
1.56
3.125
6.25
12.5
25
50
0
1.56
3.125
6.25
12.5
25
50
Compound concentration (µg/mL)
Compound concentration (µg/mL)
8f
8e
8d
8c
8b
8a
3
Doxorubicin
120
100
80
60
40
20
0
5-FU
8g
8h
8i
8j
8k
Imatinib
8l
HepG 2
120
100
80
60
40
20
0
MCF-7
0
1.56
3.125
6.25
12.5
25
50
0
1.56
3.125
6.25
12.5
25
50
Compound concentration (µg/mL)
Compound concentration (µg/mL)
Figure 2. The dose response curve showing the in vitro inhibitory activity of compounds of 8 series (8a–8l) against
(A and B) hepatocellular carcinoma (HepG2) and (C and D) breast carcinoma (MCF-7) cell lines.
Doxorubicin
120
15a
15b
15c
15d
15f
15e
Doxorubicin
120
15a
15b
15c
15d
15f
15e
HepG 2
100
80
60
40
20
0
MCF-7
100
80
60
40
20
0
0
0
5
10
15
20
25
30
35
40
45
50
1.56
3.125
6.25
12.5
25
50
Compound concentration (µg/mL)
Compound concentration (µg/m
L)
Figure 3. The dose response curve showing the in vitro inhibitory activity of compounds 15a–15f against (A) breast
carcinoma (MCF-7) and (B) hepatocellular carcinoma cell lines.
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ABBAS et al./Turk J Chem
12a
120
12b
12c
Doxorubicin
5-FU
Imatinib
12a
120
12b
12c
Doxorubicin
5-FU
Imatinib
MCF-7
HepG2
100
80
60
40
20
0
100
80
60
40
20
0
0
0.1 0.2 0.39 0.78 1.56 3.125 6.25 12.5 25
Sample conc. (µg/m
50
0
0.1 0.2 0.39 0.78 1.56 3.125 6.25 12.5 25
Sample conc. (µg/m
50
L)
L)
12a
120
12b
12c
Doxorubicin
5-FU
Imatinib
12a
120
12b
12c
Doxorubicin
5-FU
Imatinib
HCT -116
HeLa
100
80
60
40
20
0
100
80
60
40
20
0
0
0.1 0.2 0.39 0.78 1.56 3.125 6.25 12.5 25
Sample conc. (µg/m
50
0
0.1 0.2 0.39 0.78 1.56 3.125 6.25 12.5 25
Sample conc. (µg/m
50
L)
L)
Figure 4. The dose response curve showing the in vitro inhibitory activity of compounds 12a–12c compared with
reference drugs against (A) breast carcinoma (MCF-7), (B) hepatocellular carcinoma, (C) colon carcinoma, and (D)
human cervical carcinoma cell lines.
4. Experimental
4.1. Chemistry
Melting points were recorded on Gallenkamp electrothermal apparatus. 1 H NMR was determined on a Varian
Gemini 300 spectrometer (300 MHz) in DMSO-d6 with TMS as internal standard. Elemental analyses were
carried out at the Microanalytical Center, University of Cairo, Giza, Egypt. Mass spectra were recorded on a
GCMS-QP 1000 EX Shimadzu spectrometer. Infrared spectra (KBr) were determined on a Pye Unicam SP-3000
infrared spectrophotometer. Hydrazonoyl halides 543−46 were prepared according to literature procedures.
Synthesis of 3-amino-4,6-di(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (2).
A
mixture of 2-mercapto-4,6-di(4-methoxyphenyl)nicotinonitrile 1 (3.84 g, 10 mmol) and potassium hydroxide
(0.56 g, 10 mmol) in ethanol (20 mL) was refluxed for 8 h under reflux. The appropriate amount of 2-
chloroacetamide (0.93, 10 mmol) was added and stirring was continued for 2 h. The resulting solid was collected
◦
¯
and recrystallized from ethanol to give 2 as yellow crystals in 72% yield, mp 224 C; IR (KBr): V = 3428,
3328, 3263, 3173 (2NH2), 1654 (C=O), 1593 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.86 (s,
;
3H, OCH3), 3.91 (s, 3H, OCH3), 5.40 (s, D2 O exchangeable, 2H, NH2), 5.98 (s, D2 O exchangeable, 2H,
521

ABBAS et al./Turk J Chem
NH2),7.01–8.09 (m, 9H, Ar-H and pyridine-H5); MS (70 eV): m/z = 405 (M+ , 100), 180 (38), 86 (72), 58
(13). Anal. Calcd. for C22 H19 N3 O3 S (405.47): C, 65.17; H, 4.72; N, 10.36. Found: C, 65.11; H, 4.75; N,
10.18%.
Synthesis of 7,9-di(4-methoxyphenyl)-2-thioxo-2,3-dihydropyrido[3’,2’:4,5]thieno [3,2-d]pyri-
midin-4(1H )-one (3). To a stirred cold solution of 3-amino-4,6-di(4-methoxyphenyl)thieno[2,3-b]pyridine-
2-carboxamide 2 (4.05 g, 10 mmol) in 30 mL of pyridine was added 30 mL of CS2 . The mixture was heated
under reflux for 12 h and then evaporated under vacuum. The remaining product was poured into acidified
cold water, and then the solid obtained was collected by filtration, dried, and recrystallized from DMF to give
◦
¯
compound 3 as yellow crystals in 74% yield, mp 346–348 C; IR (KBr): V = 3459, 3359 (2NH), 1691 (C=O),
1600 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.84 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 7.05–8.28
;
(m, 8H, Ar-H), 7.97 (s, 1H, pyridine-H5), 9.29 (s, D2 O exchangeable, 1H, NH), 12.99 (s, D2 O exchangeable,
1H, NH); MS (70 eV): m/z = 448 (M+ + 1, 18), 447 (M+ , 100), 415 (99), 247 (22), 76 (25). Anal. Calcd.
for C23 H17 N3 O3 S2 (447.07): C, 61.73; H, 3.83; N, 9.39. Found: C, 61.54; H, 3.67; N, 9.15%.
Synthesis of 7,9-di(4-methoxyphenyl)-2-(methylthio)pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin-
4(3H )-one (4). Methyl iodide (1.42 g, 10 mmol) was added to solution of thione 3 (4.47 g, 10 mmol) in 20
mL of DMF containing anhydrous K2 CO3 (2.07 g, 15 mmol). The reaction mixture was stirred at room
temperature for 4 h and then poured onto an ice-water mixture. The solid formed was filtered, washed with
water, dried, and crystallized from DMF to give compound 4 as white crystals in 82% yield, mp 268 ◦ C; IR
−1
;
1 H NMR (300 MHz, DMSO-d6): δ = 3.56 (s, 3H,
¯
(KBr): V = 3436 (NH), 1663 (C=O), 1605 (C=N) cm
SCH3), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.05–8.24 (m, 8H, Ar-H), 7.85 (s, 1H, pyridine-H5), 12.96
(s, D2 O exchangeable, 1H, NH); MS (70 eV): m/z = 462 (M+ + 1, 14), 461 (M+ , 100), 178 (82), 151 (27),
76 (87). Anal. Calcd. for C24 H19 N3 O3 S2 (461.56): C, 62.45; H, 4.15; N, 9.10. Found: C, 62.31; H, 4.10; N,
8.87%.
4.1.1. Synthesis of 8,10-di(4-methoxyphenyl)-1,3-disubstitutedpyrido[3’,2’:4,5]thieno [3,2-d][1,2,4]
triazolo[4,3-a]pyrimidin-5(1H )-ones (8a–l).
Method A: General procedure: triethylamine (1.4 mL, 10 mmol) was added to a mixture of equimolar amounts
of thione 3 (0.447 g, 1 mmol) and the appropriate hydrazonoyl halides 5a–l (10 mmol) in 50 mL of dioxane. The
reaction mixture was refluxed until all of the starting materials disappeared and hydrogen sulfide gas ceased
to evolve (6–10 h, monitored by TLC). The solvent was evaporated and the solid that formed was filtered and
recrystallized from the appropriate solvent to give compounds 8a–l, respectively.
Method B: When the above procedure was repeated using methylthio derivative 4 (0.461 g, 1 mmol) in
lieu of 3, the product proved to be identical in all respects with 8a–l prepared above. The physical constants
of compounds 8a–l are listed below.
8,10-Di(4-methoxyphenyl)-1,3-diphenylpyrido[3’,2’:4,5]thieno[3,2-d][1,2,4] triazolo[4,3-a]
◦
¯
pyrimidin-5(1H )-one (8a). Yellow solid, mp 296–298 C (Dioxane); yield 78%; IR (KBr): V = 1693 (C=O),
1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.82 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.99–8.15
;
(m, 18H, Ar-H), 7.78 (s, 1H, pyridine-H5); 13 C NMR (300 MHz, DMSO-d6): δ = 54.6, 55.6 (2OCH3), 113.3,
114.5, 119.1, 120.4, 122.4, 124.0, 124.9, 125.1, 125.7, 127.8, 128.2, 128.4, 130.2, 131.4, 132.1, 132.4, 135.3, 135.7,
137.3, 138.7, 139.7, 146.5, 148.3, 151.7, 154.5 (Ar-C), 161.6 (C=O) ppm; MS (70 eV): m/z = 608 (M+ + 1,
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ABBAS et al./Turk J Chem
64), 607 (M+ , 100), 530 (29), 302 (26), 161 (65), 77 (73). Anal. Calcd. for C36 H25 N5 O3 S (607.68): C, 71.15;
H, 4.15; N, 11.52. Found: C, 71.05; H, 4.11; N, 11.38%.
3-Acetyl-8,10-di(4-methoxyphenyl)-1-phenylpyrido[3’,2’:4,5]thieno[3,2-d][1,2,4] triazolo[4,3-
◦
¯
a]pyrimidin-5(1H )-one (8b). Yellow solid, mp 292–294 C (DMF); yield 82%; IR (KBr): V = 1720, 1689
(2C=O), 1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 2.75 (s, 3H, COCH3), 3.76 (s, 3H, OCH3),
;
3.86 (s, 3H, OCH3), 6.99–8.19 (m, 13H, Ar-H), 7.78 (s, 1H, pyridine-H5); MS (70 eV): m/z = 574 (M+ + 1,
28), 573 (M+ , 100), 531 (14), 286 (12), 55 (23). Anal. Calcd. for C32 H23 N5 O4 S (573.62): C, 67.00; H, 4.04;
N, 12.21. Found: C, 67.13; H, 3.97; N, 12.01%.
3-Acetyl-8,10-di(4-methoxyphenyl)-1-(p-tolyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]triazolo
◦
¯
[4,3-a]pyrimidin-5(1H )-one (8c). Yellow solid, mp 304 C (DMF); yield 77%; IR (KBr): V = 1720, 1689
(2C=O), 1609 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 2.35 (s, 3H, CH3), 2.75 (s, 3H, COCH3),
;
3.83 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 7.07–8.25 (m, 12H, Ar-H), 7.84 (s, 1H, pyridine-H5); MS (70 eV):
m/z = 587 (M+ , 100), 545 (20), 91 (14), 77 (12). Anal. Calcd. for C33 H25 N5 O4 S (587.65): C, 67.45; H,
4.29; N, 11.92. Found: C, 67.27; H, 4.20; N, 11.76%.
3-Acetyl-1-(4-chlorophenyl)-8,10-di(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]
◦
¯
triazolo[4,3-a]pyrimidin-5(1H )-one (8d). Yellow solid, mp 310–312 C (DMF); yield 82%; IR (KBr): V
= 1724, 1655 (2C=O), 1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 2.75 (s, 3H, COCH3), 3.80
;
(s, 3H, OCH3), 3.91 (s, 3H, OCH3), 7.07–8.23 (m, 12H, Ar-H), 7.85 (s, 1H, pyridine-H5); MS (70 eV): m/z
= 609 (M+ + 2, 43), 607 (M+ , 100), 565 (18), 304 (14), 58 (12). Anal. Calcd. for C32 H22 ClN5 O4 S (608.07):
C, 63.21; H, 3.65; N, 11.52. Found: C, 63.19; H, 3.53; N, 11.36%.
3-Acetyl-8,10-di(4-methoxyphenyl)-1-(4-nitrophenyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]tri-
◦
¯
azolo[4,3-a]pyrimidin-5(1H )-one (8e). Brown solid, mp 348 C (DMF); yield 78%; IR (KBr): V = 1701,
1658 (2C=O), 1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 2.70 (s, 3H, COCH3), 3.83 (s, 3H,
;
OCH3), 3.88 (s, 3H, OCH3), 7.03–8.20 (m, 12H, Ar-H), 7.87 (s, 1H, pyridine-H5); MS (70 eV): m/z = 618
(M+ , 7), 401 (91), 298 (100), 69 (95). Anal. Calcd. for C32 H22 N6 O6 S (618.62): C, 62.13; H, 3.58; N, 13.59.
Found: C, 62.06; H, 3.50; N, 13.35%.
Ethyl 8,10-di(4-methoxyphenyl)-5-oxo-1-phenyl-1,5-dihydropyrido[3’,2’:4,5]thieno [3,2-d]
[1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (8f). Yellow solid, mp 262–264 ◦ C (Dioxane); yield 80%;
−1
;
1 H NMR (300 MHz, DMSO-d6): δ = 1.37 (t,J = 6.7
¯
IR (KBr): V = 1743, 1697 (2C=O), 1608 (C=N) cm
Hz, 3H, CH3 CH2), 3.81 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.43 (q, J = 6.7 Hz, 2H, CH2 CH3), 7.05–8.24
(m, 13H, Ar-H), 7.82 (s, 1H, pyridine-H5); MS (70 eV): m/z = 603 (M+ , 7), 460 (61), 359 (20), 196 (25), 92
(100). Anal. Calcd. for C33 H25 N5 O5 S (603.65): C, 65.66; H, 4.17; N, 11.60. Found: C, 65.48; H, 4.11; N,
11.43%.
Ethyl 8,10-di(4-methoxyphenyl)-5-oxo-1-(p-tolyl)-1,5-dihydropyrido[3’,2’:4,5] thieno[3,2-d]
[1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (8g). Pale yellow solid, mp 298–300 ◦ C; yield 80%; IR
−1
;
1 H NMR (300 MHz, DMSO-d6): δ = 1.38 (t, J = 6.9
¯
(KBr): V = 1751, 1693 (2C=O), 1604 (C=N) cm
Hz, 3H, CH3 CH2), 2.36 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.52 (q, J = 6.9 Hz, 2H,
CH2 CH3), 7.00–8.23 (m, 12H, Ar-H), 7.82 (s, 1H, pyridine-H5); MS (70 eV): m/z = 618 (M+ + 1, 41), 617
(M+ , 100), 544 (25), 386 (11), 91(22). Anal. Calcd. for C34 H27 N5 O5 S (617.67): C, 66.11; H, 4.41; N, 11.34.
Found: C, 71.03; H, 4.12; N, 11.42%.
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ABBAS et al./Turk J Chem
Ethyl 1,8,10-tri(4-methoxyphenyl)-5-oxo-1,5-dihydropyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]tri-
azolo[4,3-a]pyrimidine-3-carboxylate (8h). Yellow solid, mp 284–286 ◦ C (Dioxane); yield 80%; IR (KBr):
−1
;
1 H NMR (300 MHz, DMSO-d6): δ = 1.49 (t, J = 6.9 Hz, 3H,
¯
V = 1755, 1697 (2C=O), 1608 (C=N) cm
CH3 CH2), 3.70 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.58 (q, J = 6.9 Hz, 2H, CH2 CH3),
6.84–8.15 (m, 12H, Ar-H), 7.63 (s, 1H, pyridine-H5); MS (70 eV): m/z = 634 (M+ + 1, 20), 633 (M+ , 100),
561 (45), 386 (23), 148 (14), 92 (72). Anal. Calcd. for C34 H27 N5 O6 S (633.67): C, 64.44; H, 4.29; N, 11.05.
Found: C, 64.42; H, 4.13; N, 11.01%.
Ethyl 1-(4-chlorophenyl)-8,10-di(4-methoxyphenyl)-5-oxo-1,5-dihydropyrido [3’,2’:4,5]thieno
[3,2-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (8i). Pale yellow solid, mp 296 ◦ C (Dioxane); yield
−1
;
1 H NMR (300 MHz, DMSO-d6): δ = 1.41 (t,
¯
81%; IR (KBr): V = 1743, 1701 (2C=O), 1604 (C=N) cm
J = 6.7 Hz, 3H, CH3 CH2), 3.82 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.52 (q, J = 6.7 Hz, 2H, CH2 CH3),
6.93–8.17 (m, 12H, Ar-H), 7.76 (s, 1H, pyridine-H5); MS (70 eV): m/z = 640 (M+ + 2, 2), 638 (M+ , 7), 415
(31), 196 (44), 105 (10), 92 (100), 65 (23). Anal. Calcd. for C33 H24 ClN5O5S (638.09): C, 62.12; H, 3.79; N,
10.98. Found: C, 62.08; H, 3.70; N, 10.76%.
8,10-Di(4-methoxyphenyl)-5-oxo-N,1-diphenyl-1,5-dihydropyrido[3’,2’:4,5]thieno [3,2-d]
[1,2,4]triazolo[4,3-a]pyrimidine-3-carboxamide (8j). Yellowish white solid, mp 348 ◦ C (DMF); yield 80%;
−1
;
1 H NMR (300 MHz, DMSO-d6): δ =
¯
IR (KBr): V = 3444 (NH), 1691, 1674 (2C=O), 1603 (C=N) cm
3.82 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 7.02–8.25 (m, 18H, Ar-H), 7.86 (s, 1H, pyridine-H5), 11.30 (s, D2 O
exchangeable, 1H, NH); MS (70 eV): m/z = 650 (M+ , 18), 447 (49), 195 (38), 92 (100). Anal. Calcd. for
C37 H26 N6 O4 S (650.71): C, 68.29; H, 4.03; N, 12.92. Found: C, 68.13; H, 4.01; N, 12.76%.
8,10-Di(4-methoxyphenyl)-5-oxo-N-phenyl-1-(p-tolyl)-1,5-dihydropyrido[3’,2’:4,5] thieno[3,2-
d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxamide (8k). Yellowish white solid, mp 353–355 ◦ C (DMF);
−1
;
1 H NMR (300 MHz, DMSO-
¯
yield 84%; IR (KBr): V = 3448 (NH), 1687, 1670 (2C=O), 1603 (C=N) cm
d6): δ = 2.36 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 7.02–8.25 (m, 17H, Ar-H), 7.86 (s, 1H,
pyridine-H5), 11.30 (s, D2 O exchangeable, 1H, NH); MS (70 eV): m/z = 665 (M+ + 1, 85), 664 (M+ , 71),
426 (100), 311 (98), 175 (86), 56 (79). Anal. Calcd. for C38 H28 N6 O4 S (664.73): C, 68.66; H, 4.25; N, 12.64.
Found: C, 68.43; H, 4.15; N, 12.39%.
1-(4-Chlorophenyl)-8,10-di(4-methoxyphenyl)-5-oxo-N-phenyl-1,5-dihydropyrido [3’,2’:4,5]
thieno[3,2-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxamide (8l). Yellowish-white solid, mp 336–338 ◦ C
−1
;
1 H NMR (300 MHz,
¯
(DMF); yield 88%; IR (KBr): V = 3436 (NH), 1689, 1673 (2C=O), 1601 (C=N) cm
DMSO-d6): δ = 3.82 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 7.02–8.25 (m, 17H, Ar-H), 7.88 (s, 1H, pyridine-H5),
11.28 (s, D2 O exchangeable, 1H, NH); MS (70 eV): m/z = 687 (M+ + 2, 11), 685 (M+ , 54), 553 (18), 421
(93), 323 (100), 152 (94), 78 (93), 65 (51). Anal. Calcd. for C37 H25 ClN6 O4 S (685.15): C, 64.86; H, 3.68; N,
12.27. Found: C, 64.59; H, 3.48; N, 12.05%.
4.1.2. Synthesis of 12a–c
General procedure: 1 mL of an aqueous solution of KOH (75%) was added to 4.47 g of 3 (10 mmol) in 50
mL of ethanol and the mixture was warmed for 10 min. The appropriate chloromethylene compound 11a–c
(10 mmol) was added to the resulting clear solution and then the reaction mixture was stirred for 12 h at room
temperature. The solid that precipitated was filtered off, washed with water, dried, and crystallized from the
appropriate solvent to give 12a–c, respectively.
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ABBAS et al./Turk J Chem
3-((7,9-Di(4-methoxyphenyl)-4-oxo-3,4-dihydropyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin-2-yl)
◦
¯
thio)pentane-2,4-dione (12a).Yellowish white solid, mp 210–212 C (Dioxane); yield 86%; IR (KBr): V =
3444 (NH), 1683, 1651, 1643 (3C=O), 1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 2.34 (s, 6H,
;
2COCH3), 3.88 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.01 (s, 1H, CH), 6.97–8.16 (m, 8H, Ar-H), 7.26 (s, 1H,
pyridine-H5), 12.18 (s, D2 O exchangeable, 1H, NH); MS (70 eV): m/z = 545 (M+ , 2), 529 (100), 448 (55),
328 (15), 136 (15), 71 (7). Anal. Calcd. for C28 H23 N3 O5 S2 (545.63): C, 61.64; H, 4.25; N, 7.70. Found: C,
61.61; H, 4.17; N, 7.53%.
Ethyl 2-((7,9-di(4-methoxyphenyl)-4-oxo-3,4-dihydropyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin
-2-yl)thio)-3-oxobutanoate (12b). Yellowish white solid, mp 180–182 ◦ C (Dioxane/EtOH); yield 73%; IR
−1
;
1 H NMR (300 MHz, DMSO-d6): δ
¯
(KBr): V = 3417 (NH), 1739, 1655, 1641 (3C=O), 1608 (C=N) cm
= 1.35 (t, J = 7.2 Hz, 3H, CH3 CH2), 2.09 (s, 3H, COCH3), 3.89 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.82
(s, 1H, CH), 4.34 (q, J = 7.2 Hz, 2H, CH2 CH3), 6.96–8.16 (m, 9H, Ar-H and pyridine-H5), 12.04 (s, D2 O
exchangeable, 1H, NH), MS (70 eV): m/z = 575 (M+ , 7), 486 (100), 429 (98), 316 (78), 213 (39), 136 (43),
65 (45). Anal. Calcd. for C29 H25 N3 O6 S2 (575.66): C, 60.51; H, 4.38; N, 7.30. Found: C, 60.38; H, 4.33; N,
7.15%.
2-((7,9-Di(4-methoxyphenyl)-4-oxo-3,4-dihydropyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin-2-yl)
thio)-3-oxo-N-phenylbutanamide (12c). Yellowish white solid, mp 202–204 ◦ C (Dioxane); yield 79%; IR
−1
;
1 H NMR (300 MHz, DMSO-d6):
¯
(KBr): V = 3412, 3363 (2NH), 1692, 1659, 1648 (3C=O), 1602 (C=N) cm
δ = 2.10 (s, 3H, COCH3), 3.84 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.03 (s, 1H, CH), 6.78–8.15 (m, 14H,
Ar-H and pyridine-H5), 8.84 (s, D2 O exchangeable, 1H, NH), 10.39 (s, D2 O exchangeable, 1H, NH); MS (70
eV): m/z = 622 (M+ , 7), 617 (100), 441 (43), 262 (79), 164 (21), 59 (90). Anal. Calcd. for C33 H26 N4 O5 S2
(622.71): C, 63.65; H, 4.21; N, 9.00. Found: C, 63.45; H, 4.13; N, 8.87%.
4.1.3. Alternate synthesis of 8b, 8f, and 8j
A cold solution of benzenediazonium chloride, prepared by diazotizing aniline (1 mmol) dissolved in hydrochloric
acid (6 M, 1 mL) with a solution of sodium nitrite (0.07 g, 1 mmol) in water (2 mL) was added portion-wise to
a solution of the appropriate 12a–c (1 mmol) in ethanol (20 mL) containing sodium acetate trihydrate (0.138
g, 1 mmol) while stirring and keeping the temperature below 5 ◦ C. The reaction mixture was left for 3 h in
a refrigerator. The solid precipitated was filtered off, washed with water, dried, and crystallized from DMF to
give compounds 8b, 8f, and 8j, which were identical in all respects (mp, mixed mp, and IR spectra) with those
obtained from reaction of 3 with 5b, 5f, and 5j, respectively.
Synthesis of 2-hydrazinyl-7,9-di(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin-
4(3H )-one (13). Hydrazine hydrate (80%, 20 mL) was added to 7,9-di(4-methoxyphenyl)-2-(methylthio)pyrido
[3’,2’:4,5]thieno[3,2-d]pyrimidin-4(3H)-one 4 (4.61 g, 10 mmol) in dry EtOH (40 mL) and the reaction mixture
was kept under reflux for 10 h and then cooled. The solid that precipitated was filtered off and crystallized
◦
¯
from DMF to give 13 as canary yellow solid, 70% yield, mp 320–322 C; IR (KBr): V = 3448, 3348, 3301,
3220 (NH2 and 2NH), 1678 (C=O), 1652 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 1.90 (s, D2 O
;
exchangeable, 1H, NH), 3.36 (s, D2 O exchangeable, 2H, NH2), 3.73 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
7.02–8.22 (m, 8H, Ar-H), 7.76 (s, 1H, pyridine-H5), 8.12 (s, D2 O exchangeable, 1H, NH); MS (70 eV): m/z =
445 (M+ , 7), 415 (7), 302 (4), 196 (64), 92 (100), 66 (31); Anal. Calcd. for C23 H19 N5 O3 S (445.49): C, 62.01;
H, 4.30; N, 15.72%. Found: C, 62.24; H, 4.13; N, 15.48%.
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4.1.4. Synthesis of hydrazones 15a–f
A mixture of hydrazine 13 (0.89 g, 2 mmol) and the appropriate aldehyde 14a–f (2 mmol) in acetic acid (20
mL) and a few drops of conc. hydrochloric acid (≈1 mL) was heated under reflux for 5 h. The reaction mixture
was then cooled and diluted with water. The so-formed solid product was then collected by filtration, dried,
and recrystallized from DMF to afford the corresponding hydrazones 15a–f.
2-(2-Benzylidenehydrazinyl)-7,9-di(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d] pyrimidin-
◦
¯
4(3H )-one (15a). Yellow solid, mp 346–348 C; yield 74%; IR (KBr): V = 3444, 3359 (2NH), 1658 (C=O),
1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.84 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 7.06–8.25
;
(m, 13H, Ar-H), 7.92 (s, 1H, pyridine-H5), 8.17 (s, 1H, =CH), 11.11, 11.79 (2s, D2 O exchangeable, 2H, 2NH);
MS (70 eV): m/z = 533 (M+ , 61), 430 (54), 387 (26), 301 (14), 228 (12),104 (100), 77 (69). Anal. Calcd. for
C30 H23 N5 O3 S (533.60) C, 67.53; H, 4.34; N, 13.12. Found: C, 67.39; H, 4.37; N, 13.04%.
2-(2-(4-Chlorobenzylidene)hydrazinyl)-7,9-di(4-methoxyphenyl)pyrido[3’,2’:4,5] thieno[3,2-
◦
¯
d]pyrimidin-4(3H )-one (15b). Yellow solid, mp 352–354 C; yield 73%; IR (KBr): V = 3448, 3355
(2NH), 1651 (C=O), 1604 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.82 (s, 3H, OCH3), 3.88 (s,
;
3H, OCH3), 7.04–8.22 (m, 12H, Ar-H), 7.78 (s, 1H, pyridine-H5), 8.13 (s, 1H, =CH), 11.16, 11.86 (2s, D2 O
exchangeable, 2H, 2NH); MS (70 eV): m/z = 569 (M+ + 2, 20), 567 (M+ , 38), 456 (50), 359 (19), 275 (16),
137 (64), 104 (12), 73 (100). Anal. Calcd. for C30 H22 ClN5 O3 S (568.05) C, 63.43; H, 3.90; N, 12.33. Found:
C, 63.26; H, 3.76; N, 12.16%.
2-(2-(4-Methoxybenzylidene)hydrazinyl)-7,9-bis(4-methoxyphenyl)pyrido[3’,2’:4,5] thieno
◦
¯
[3,2-d]pyrimidin-4(3H )-one (15c). Yellow solid, mp 324–326 C; yield 76%; IR (KBr): V = 3444, 3348
(2NH), 1662 (C=O), 1604 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.80 (s, 3H, OCH3), 3.85 (s,
;
3H, OCH3), 3.89 (s, 3H, OCH3), 6.95–8.25 (m, 12H, Ar-H), 7.80 (s, 1H, pyridine-H5), 8.11 (s, 1H, =CH),
10.98, 11.69 (2s, D2 O exchangeable, 2H, 2NH); MS (70 eV): m/z = 563 (M+ , 18), 415 (100), 359 (24), 273
(14), 135 (26), 99 (16), 75 (16). Anal. Calcd. for C31 H25 N5 O4 S (563.63) C, 66.06; H, 4.47; N, 12.43. Found:
C, 66.25; H, 4.30; N, 12.23%.
7,9-Di(4-methoxyphenyl)-2-(2-(4-nitrobenzylidene)hydrazinyl)pyrido[3’,2’:4,5] thieno[3,2-d]
◦
¯
pyrimidin-4(3H )-one (15d). Orange solid, mp 350 C; yield 73%; IR (KBr): V = 3448, 3363 (2NH), 1670
(C=O), 1604 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.81 (s, 3H, OCH3), 3.88 (s, 3H, OCH3),
;
6.98–8.23 (m, 12H, Ar-H), 7.86 (s, 1H, pyridine-H5), 8.19 (s, 1H, =CH), 11.08, 11.81 (2s, D2 O exchangeable,
2H, 2NH); MS (70 eV): m/z = 578 (M+ , 1), 447 (79), 330 (6), 287 (43), 121 (66), 92 (32), 58 (100). Anal.
Calcd. for C30 H22 N6 O5 S (578.60) C, 62.27; H, 3.83; N, 14.52. Found: C, 62.20; H, 3.64; N, 14.31%.
2-(2-(2-Hydroxybenzylidene)hydrazinyl)-7,9-bis(4-methoxyphenyl)pyrido[3’,2’:4,5] thieno
◦
¯
[3,2-d]pyrimidin-4(3H )-one (15e). Yellow solid, mp 331–333 C; yield 74%; IR (KBr): V = 3506–3444,
3375 (OH and 2NH), 1658 (C=O), 1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.84 (s, 3H,
;
OCH3), 3.88 (s, 3H, OCH3), 6.84–8.24 (m, 12H, Ar-H), 7.80 (s, 1H, pyridine-H5), 8.49 (s, 1H, =CH), 9.88,
11.12 (2s, D2 O exchangeable, 2H, 2NH), 11.90 (1s, 1H, D2 O exchangeable, OH); MS (70 eV): m/z = 549
(M+ , 17), 359 (19), 274 (8), 120 (44), 85 (98), 57 (100). Anal. Calcd. for C30 H23 N5 O4 S (549.60) C, 65.56;
H, 4.22; N, 12.74. Found: C, 65.50; H, 4.16; N, 12.53%.
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2-(2-(2,4-Dichlorobenzylidene)hydrazinyl)-7,9-di(4-methoxyphenyl)pyrido[3’,2’:4,5 ]thieno
◦
¯
[3,2-d]pyrimidin-4(3H)-one (15f). Yellow solid, mp 344–345 C; yield 78%; IR (KBr): V = 3448, 3348
(2NH), 1662 (C=O), 1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.78 (s, 3H, OCH3), 3.87 (s,
;
3H, OCH3), 7.18–8.24 (m, 11H, Ar-H), 7.88 (s, 1H, pyridine-H5), 8.20 (s, 1H, =CH), 11.13, 11.89 (2s, D2 O
exchangeable, 2H, 2NH); MS (70 eV): m/z = 603 (M+ , 2), 447 (100), 430 (45), 315 (39), 121 (47), 59 (77).
Anal. Calcd. for C30 H21 Cl2 N5 O3 S (602.49) C, 59.81; H, 3.51; N, 11.62. Found: C, 59.79; H, 3.48; N, 11.50%.
4.1.5. Cyclization of hydrazones 15a–f
Bromine (0.052 g, 1 mmol) in acetic acid (5 mL) was added dropwise to a stirred solution of the appropriate
hydrazone 15a–f (1 mmol of each) in acetic acid (10 mL) and sodium acetate (0.5 g). The reaction mixture was
then poured onto ice cold water, and the solid that precipitated was filtered off, washed with sodium bicarbonate
solution and then with water, dried, and crystallized from DMF to give the respective compounds 16a–f.
8,10-Di(4-methoxyphenyl)-3-phenylpyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]triazolo [4,3-a] pyri=
◦
¯
midin-5(1H )-one (16a). White solid, mp 320 C; yield 69%; IR (KBr): V = 3434 (NH), 1689 (C=O), 1608
(C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.82 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.63–8.28 (m,
;
13H, Ar-H), 7.84 (s, 1H, pyridine-H5), 11.76 (s, D2 O exchangeable, 1H, NH); 13 C NMR (300 MHz, DMSO-d6):
δ = 54.3, 55.2 (2OCH3), 114.9, 117.5, 119.1, 122.1, 122.6, 124.9, 125.1, 125.7, 127.8, 129.1, 131.4, 132.1, 133.7,
135.3, 135.7, 137.3, 138.7, 139.9, 153.6, 154.1, 156.5 (Ar-C), 162.0 (C=O) ppm; MS (70 eV): m/z = 531 (M+ ,
26), 432 (100), 415 (92), 388 (27), 273 (6), 180 (43), 64 (59). Anal. Calcd. for C30 H21 N5 O3 S (531.58) C,
67.78; H, 3.98; N, 13.17. Found: C, 67.57; H, 3.79; N, 13.03%.
3-(4-Chlorophenyl)-8,10-di(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4] triazolo[4,3-
◦
¯
a]pyrimidin-5(1H )-one (16b). Yellow solid, mp 330–332 C; yield 68%; IR (KBr): V = 3432 (NH), 1688
(C=O), 1608 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.83 (s, 3H, OCH3), 3.90 (s, 3H, OCH3),
;
7.12–8.28 (m, 12H, Ar-H), 7.83 (s, 1H, pyridine-H5), 11.87 (s, D2 O exchangeable, 1H, NH); MS (70 eV): m/z
= 568 (M+ + 2, 31), 566 (M+ , 98), 432 (7), 398 (20), 250 (9), 120 (40), 92 (41), 64 (100). Anal. Calcd. for
C30 H20 ClN5 O3 S (566.03) C, 63.66; H, 3.56; N, 12.37. Found: C, 63.51; H, 3.44; N, 12.25%.
3,8,10-Tri(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin -5
◦
¯
(1H )-one (16c). White solid, mp 318–319 C; yield 68%; IR (KBr): V = 3433 (NH), 1686 (C=O), 1607
(C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.78 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.90 (s, 3H,
;
OCH3), 6.74–8.29 (m, 12H, Ar-H), 7.89 (s, 1H, pyridine-H5), 11.67 (s, D2 O exchangeable, 1H, NH); MS (70
eV): m/z = 561 (M+ , 4), 432 (100), 415 (94), 388 (26), 135 (21), 64 (67). Anal. Calcd. for C31 H23 N5 O4 S
(561.61) C, 66.30; H, 4.13; N, 12.47. Found: C, 66.16; H, 4.02; N, 12.40%.
8,10-Di(4-methoxyphenyl)-3-(4-nitrophenyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4] triazolo[4,3-
◦
¯
a]pyrimidin-5(1H )-one (16d). Brown solid, mp 347–349 C; yield 71%; IR (KBr): V = 3434 (NH), 1693
(C=O), 1606 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.79 (s, 3H, OCH3), 3.85 (s, 3H, OCH3),
;
6.72–8.28 (m, 12H, Ar-H), 7.81 (s, 1H, pyridine-H5), 11.82 (s, D2 O exchangeable, 1H, NH); MS (70 eV): m/z
= 576 (M+ , 5), 432 (57), 388 (6), 135 (11), 92 (12), 64 (100). Anal. Calcd. for C30 H20 N6 O5 S (576.58) C,
62.49; H, 3.50; N, 14.58. Found: C, 62.42; H, 3.31; N, 14.46%.
3-(2-Hydroxyphenyl)-8,10-di(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4] triazolo
◦
¯
[4,3-a]pyrimidin-5(1H )-one (16e). Yellow solid, mp 352–353 C; yield 71%; IR (KBr): V = 3519–3400
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ABBAS et al./Turk J Chem
(OH and NH), 1673 (C=O), 1606 (C=N) cm−1
;
1 H NMR (300 MHz, DMSO-d6): δ = 3.80 (s, 3H, OCH3),
3.85 (s, 3H, OCH3), 6.68–8.24 (m, 12H, Ar-H), 7.81 (s, 1H, pyridine-H5), 11.12 (s, D2 O exchangeable, 1H,
NH), 11.83 (s, 1H, D2 O exchangeable, OH); MS (70 eV): m/z = 547 (M+ , 3), 432 (100), 388 (24), 137 (15),
60 (91). Anal. Calcd. for C30 H21 N5 O4 S (547.58) C, 65.80; H, 3.87; N, 12.79. Found: C, 65.59; H, 3.81; N,
12.58%.
3-(2,4-Dichlorophenyl)-8,10-di(4-methoxyphenyl)pyrido[3’,2’:4,5]thieno[3,2-d][1,2,4]triazolo
◦
¯
[4,3-a]pyrimidin-5(1H )-one (16f). White solid, mp 316–318 C; yield 72%; IR (KBr): V = 3433 (NH),
1691 (C=O), 1605 (C=N) cm−1 1 H NMR (300 MHz, DMSO-d6): δ = 3.80 (s, 3H, OCH3), 3.98 (s, 3H,
;
OCH3), 6.67–8.33 (m, 11H, Ar-H), 7.89 (s, 1H, pyridine-H5), 11.74 (s, D2 O exchangeable, 1H, NH); MS (70
eV): m/z = 601 (M+ , 44), 494 (48), 432 (100), 387 (29), 136 (45), 64 (76). Anal. Calcd. for C30 H19 Cl2 N5 O3 S
(600.47) C, 60.01; H, 3.19; N, 11.66. Found: C, 59.89; H, 3.04; N, 11.47%.
4.1.6. Alternate synthesis of 16a
To a solution of hydrazine 13 (0.445 g, 1 mmol) in dry pyridine (20 mL) was added benzoyl chloride (0.14 g, 1
mmol) and the resulting mixture was refluxed for 2 h. After cooling, the precipitate was collected by filtration
and crystallized from DMF to afford a product that was found to be identical in all respects (mp, mixed mp,
and IR) with product 16a.
4.2. Biological evaluation
4.2.1. Antimicrobial activity assay
All microbial strains were provided from the culture collection of the Regional Center for Mycology and Biotech-
nology (RCMB), Al-Azhar University, Cairo, Egypt. The preliminary antimicrobial activity was investigated on
a dozen newly synthesized compounds in order to increase the selectivity of these derivatives towards test mi-
croorganisms. Briefly, 100 µL of the test bacteria/fungi were grown in 10 mL of fresh media until they reached
a count of approximately 108 cells/mL for bacteria or 105 cells/mL for fungi.47 Then 100 µL of microbial
suspension was spread onto agar plates corresponding to the broth in which they were maintained and tested
for susceptibility by the well diffusion method. The National Committee for Clinical Laboratory Standards
(NCCLS) recommends Mueller-Hinton and Sabouraud agar as they result in good batch-to-batch reproducibil-
ity. The diffusion method for filamentous fungi and yeast was tested by using the approved standard methods
(M38-A and M44-P, respectively) developed by the NCCLS48 for evaluating susceptibilities to antifungal agents.
One hundred microliters of each sample (at 5 mg/mL) was added to each well (10 mm diameter holes cut in the
agar gel). The plates were incubated for 24–48 h at 37 ◦ C (for bacteria and yeast) and for 48 h at 28 ◦ C (for
filamentous fungi). After incubation, the microorganism’s growth was observed. The resulting inhibition zone
diameters were measured in millimeters and used as the criterion for antimicrobial activity. If an organism is
placed on the agar it will not grow in the area around the well if it is susceptible to the chemical. This area
of no growth around the disk is known as a zone of inhibition. The size of the clear zone is proportional to
the inhibitory action of the compound under investigation. Solvent controls (DMSO) were included in every
experiment as negative controls. DMSO was used for dissolving the tested compounds and showed no inhibition
zones, confirming that it has no influence on growth of the tested microorganisms.
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4.2.2. MIC determination using the broth microdilution method
The in vitro antimicrobial activity of the synthesized compounds was screened using the broth dilution method
as described by CLSI49 to determine the lowest concentration inhibiting growth of the organism recorded as the
MIC using DMSO as diluent. Mueller-Hinton broth was used as nutrient medium to grow and dilute the drug
suspension for the tested bacteria, and Sabouraud dextrose broth was used for fungal nutrition. The stock 1000
µg/mL was prepared. For the broth microdilution test, 50 µL of each microbial suspension in suitable growth
medium was added to the wells of a sterile 96-well microtiter plate already containing 50 µL of two-fold serially
diluted tested compound. Control wells were prepared with culture medium, microbial suspension only, tested
compound only, and DMSO in amounts corresponding to the highest quantity present. The contents of each
well were mixed on a microplate shaker at 900 rpm for 1 min prior to incubation for 24–48 h in the cultivation
conditions described above. The MIC was the lowest concentration where no viability was observed after 24–48
h on the basis of metabolic activity. To indicate respiratory activity the presence of color was determined
after adding 10 µL/well of 2,3,5-triphenyl tetrazolium chloride dissolved in water (20 mg/mL) and incubation
under appropriate cultivation conditions for 30 min in the dark.50 After incubation, the optical density was
measured by a microplate reader. Positive controls were wells with a microbial suspension in an appropriate
growth medium in amounts corresponding to the highest quantity present in the broth microdilution assay.
Negative controls were wells with growth medium and tested compound. All measurements of MIC values were
repeated in triplicate. Tetracycline and ampicillin were used as standard antibacterial drugs, while griseofulvin
and amphotericin B were used as standard antifungal drugs.
4.2.3. Evaluation of the antitumor activity using a viability assay
All human anticancer cell lines were obtained from the American Type Culture Collection. The cells were grown
on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamicin. The
cells were maintained at 37 ◦ C in a humidified atmosphere with 5% CO2 and were subcultured two to three
times a week.
Gangadevi and Muthumary’s method51 was used for evaluation of the potential cytotoxicity of the tested
compounds. The number of surviving cells was determined by staining the cells with crystal violet51,52 followed
by cell lysing using 33% glacial acetic acid and reading the absorbance at 590 nm using a microplate reader
(SunRise, TECAN, Inc, USA) after mixing well.
The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of
intact cells, was estimated from graphic plots.
5. Conclusion
A novel series of fused [1,2,4]triazolo[1,5-a]pyrimidines were synthesized by different methods and evaluated for
their in vitro antibacterial, antifungal, and anticancer activities. From the screening results, it can be seen that
compounds 8f and 15f showed excellent activity against gram-positive bacteria and compound 15d showed
potent activity against gram-negative bacteria. The rest of the compounds showed good or moderate activity
against the tested bacteria compared with the standard drugs. Compounds 8d, 8e, 8c, 8l, and 8j among
the compounds tested exhibited higher antifungal activity as compared with the reference drug against yeast
species. The in vitro cytotoxic activity testing revealed that compounds 12c, 12a, and 12b exhibited promising
inhibitory activity against the four tested tumor cells (MCF-7, HepG-2, HCT-116, and HeLa).
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